Outline Terminology - University of Minnesota · PDF file•Detergent and salt free solution ... pigments were passed through calcium carbonate using petroleum ether ... Nano LC =

Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279

Outline

• Electrospray

• Protein ESI-MS

• Peptide Separation

• MS Acquisition

• Peptide Fragmentation

Terminology

• ESI

• Charge state

• Isotope series

• Monoisotopic

• Liquid chromatography

• MS1 and MS2

• Precursor ion

• MS2 (same as: MS/MS, Tandem MS or Product ion spectrum)

• Product Ions

• b-ion, y-ion


Gingras A et al, 2007 Nat Rev Mol Cell Biol 8, 645

A. Obtain protein-complex B. Separate proteins on 1D gel C. Excise protein bands > trypsin digestion D. Liquid Chromatography-ESI-Mass Spec Analysis of

peptide mixtures

SDS-PAGE

EXAMPLE 1 Workflow: Protein Complex Analysis with Mass Spectrometry


Electrospray Ionization (ESI) Formation of and/or Transfer of charged ions from solution phase to gas phase

Metz TO, et al. (2007) Biomarkers Med 1(1), 159-185

Solution phase

Gas phase

measure m/z in mass spectrometer


Electrospray Ionization “Method of generating a very fine liquid aerosol through electrostatic charging”

http://www.newobjective.com/electrospray/index.html

1. Droplets (<10 µm diameter) are generated by electrically charging a liquid to a very high voltage 2. Droplets rapidly shrink as solvent molecules evaporate from their surface 3. Charges become close in proximity 4. Charge repulsion occurs 5. Droplets explode, charges are pulled towards counter electrode, into MS

• “Soft“ ionization since the molecule being ionized does not fall apart or break-up during the process • First ESI publication: Zeleny, J. The Physical Review 3 (1914):69-91 • John Fenn: 2002 Chemistry Nobel Prize- joint recipient for electrospray

Adapted from http://www.newobjective.com/electrospray/index.html

SUPPLEMENTAL: ESI Reference Slide


Electrospray Formation of and/or Transfer of ions from solution phase to gas phase

Ref: IJAC Vol 2012 ID 282574

Spray Nozzle/Needle Mass Spectrometer

SUPPLEMENTAL: ESI Reference Slide


Electrospray Ionization (ESI)

• Peptide ISOTOPE series • Protein CHARGE series


Mass Spectrometry Data

𝑚

𝑧 =

𝑚𝑎𝑠𝑠

𝑐ℎ𝑎𝑟𝑔𝑒


Peptide AVDVLQQMPLK

NH3+

COOH

[AVDVLQQMPLK]+1H

C55H97N14O16S

𝑚

𝑧=

1241.693

1

1241.692

AVDVLQQMPLK

C55H96N14O16S

m = 1240.685

Neutral peptide

Singly charged peptide [M + H]+1

NH2

COOH

(Numbers are monoisotopic)


[AVDVLQQMPLK]+1H

C55H97N14O16S

1241.692

[AVDVLQQMPLK ]+1H

C5413CH97N14O16S

1242.696 + 1.00335

[AVDVLQQMPLK ]+1H

C5313C2H97N14O16S

1243.699 + 1.00335

12C molar mass = 12.00000 13C molar mass = 13.00335

Isotopically Related Singly Charged Peptide Species


higgi022_20140703_13837_c_ellie_13to19_MS1_prof #10148-10248 RT: 75.89-76.64 AV: 101 NL: 1.03E5T: FTMS + p NSI Full ms [360.00-1800.00]

1239 1240 1241 1242 1243 1244 1245 1246

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative A

bundance

1241.692z=1

1242.695z=1

1243.698z=1

1.003

1.003

Isotopically Related Singly Charged Peptide Species AVDVLQQMPLK

[AVDVLQQMPLK]+1H

C55H96N14O16S

[AVDVLQQMPLK]+1H

C5413CH96N14O16S

[AVDVLQQMPLK]+1H

C5313C2H96N14O16S

Monoisotopic peak (Definition: a peak containing only ions made up of the principal isotopes of atoms making up the original molecule. (IUPAC))

Stable Isotopes: 13C, 15N, 18O, 34S etc


NH3+

COOH

H+

[AVDVLQQMPLK]+1H

C55H98N14O16S

𝑚

𝑧=

1242.701

2

621.351

Doubly charged peptide [M + 2H]2+

monoisotopic

Mass spectrum signal


[AVDVLQQMPLK]+1H

C55H97N14O16S

1241.692

[AVDVLQQMPLK]+1H

C5413CH97N14O16S

1242.695

[AVDVLQQMPLK]+1H

C5313C2H97N14O16S

1243.699

Isotopically Related Singly Charged Peptide Species

[AVDVLQQMPLK]+2H

C55H98N14O16S

𝑚

𝑧=

1242.701

2

621.351

[AVDVLQQMPLK]+2H

C5413CH98N14O16S

𝑚

𝑧=

1243.704

2

621.852

0.5

Isotopically Related Doubly Charged Peptide Species

0.5

+ 1.00335 + 1.00335

[AVDVLQQMPLK]+2H

C5313C2H98N14O16S

𝑚

𝑧=

1244.707

2

622.354


higgi022_20140703_13837_c_ellie_13to19_MS1_prof #10148-10248 RT: 75.89-76.64 AV: 101 NL: 1.70E6T: FTMS + p NSI Full ms [360.00-1800.00]

620.0 620.5 621.0 621.5 622.0 622.5 623.0 623.5

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative A

bundance

621.350z=2

621.851z=2

622.352z=2

0.5 0.5

Isotopically Related Doubly Charged Peptide Species: AVDVLQQMPLK

1) Monoisotopic peak

2) Doubly charged species: 1

0.5 = 2

the mass difference between adjacent isotopically-related peaks is used to calculate the charge state


Finding peptides in data:

• Monoisotopic mass

• Spacing between isotopic peaks (provides charge state)

• Shape of the isotopic envelope (relative intensities of peaks)


Intact Protein Electrospray - Mass Spectrum

200 1000 2000 m/z

+7 1766.6

+8 1545.7

+9 1374.2

+10 1236.9

+11 1124.6

+12 1031.0

+13 951.8

+14 884.0

+15 825.0

+16 772.4

+17 727.5

0

50

100

Rel

ativ

e A

bu

nd

ance

PROTEIN CHARGE SERIES

10000

mass

12500 15000 0

50

100

Rel

ativ

e A

bu

nd

ance

12359 +/- 2

Mathematically deconvoluted Data Protein MW


Intact Protein ESI-MS Cytochrome C, 12361 Da

m

z =

12361 + 8

8 = 1546

+8

m

z =

12361 + 12

12 = 1031

+12

1031.0

200 1000 2000 m/z

1545.7

0

50

100

Re

lati

ve A

bu

nd

an

ce

NH3+

COOH

H+

H+

H+ H+

H+ H+ H+

Charge = +8


Intact Protein Electrospray-MS Infusion of Solubilized Relatively Pure Protein

• Relatively pure sample • 20 – 50 M protein concentration • Detergent and salt free solution • Typical solvent: 50:50, acetonitrile:water, 0.1% formic

acid • Difficult (but possible) to achieve high quality data • Non-covalent interactions are retained with ESI (not

MALDI), usually with neutral/basic buffer system and a lot of trial and error


+TOF MS: 65 MCA scans fro...a=3.57017668630689880e-00...

Max. 8177.0 counts.

1600 1800 2000 2200m/z, amu

0

2000

4000

6000

8000

Inte

ns

ity

, c

ou

nts

17921730

1858

1672

1930

1618 2007

18151753 209018821568

m/z

Inte

nsi

ty, c

ou

nts

Intermediate 1 Protein

Glycosylated Intact Protein ESI Mass Spec


Intermediate 1: Deconvoluted MS Data Using Bayesian Reconstruct Tool; Theoretical mass = 50140.01 Da; error=4 ppm

Mass reconstruction of +TOF MS: ...a=3.57017668630689880e-004, t0...

Max. 1.1e5 cps.

5.00e4 5.05e4 5.10e4Mass, amu

0.00

2.00e4

4.00e4

6.00e4

8.00e4

1.00e5

1.13e5

Inte

ns

ity

, c

ps

50140

50180

50797502295049749849

Inte

nsi

ty, c

ps

1.0e5

8.0e4

6.0e4

4.0e4

2.0e4

0

50943

.23

656

+K

+K

Sialic acid

291

Fucose

146

NAcNeu|

Gal|

GlcNAc


Complex Samples Cannot be Infused Directly into the Mass Spectrometer

Mixtures are separated or partially separated before MS analysis with

Chromatographic Technologies


Peptide Separation:

Liquid Chromatography-Mass Spectrometry (LC-MS) Analyte mixtures are separated or partially separated before MS analysis with

column chromatography

http://www.chemistry.adelaide.edu.au/external/soc-rel/content/lc-col.htm

“… modern Liquid Chromatography (LC), uses a liquid mobile phase to transport the sample components through a column packed with a solid material - the stationary phase.” Reference: http://www.earl2learn.com

From Tswett’s notebook (1910) on the early chromatographic experiments: plant pigments were passed through calcium carbonate using petroleum ether

Mikhail Tswett (1872 – 1919)


higgi022_091711_sPRG_pep_digestmix_HC... 9/17/2011 11:27:17 PM

RT: 0.00 - 70.01

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

41.21

36.2726.13

38.809.43

31.80

24.56

33.72

19.7028.01 48.65

18.35 20.49

44.71

30.98

17.65 44.87

55.2515.6450.56

57.679.0663.0352.05 58.728.53 67.1212.712.57

NL: 1.63E8

Base Peak F: FTMS + c NSI Full ms [300.00-1800.00] MS higgi022_091711_sPRG_pep_digestmix_HCD_40_01

• Peptide Elution Profile

• Increased Retention Time = increased peptide hydrophobicity

1 Dimensional LC-MS Elution Profile


Peptide Separation with C18 Stationary Phase Hydrophobic Functional Group = Octadecylsilyl or “C18”

Si

O

SiCH3

3HC

Si

O

Si CH3

3HC

Si

O

Si CH33HC

Si

O

Si CH33HC

Si

O

Si CH3

3HC

Peptides bind to C18 functional groups (carbon-18 chains) with hydrophobic interactions

5 um

(not drawn to scale: C18 groups are in pores (e.g., 200 Å) inside the silica)


HPLC Column Parameters (High Performance/Pressure Liquid Chromatography)

Category Column Internal

Diameter Column flow rate range

Capillary

Capillary at MSP

50 – 300 um

75 & 100 um

~0.05 – 20 ul/min

~0.3 ul/min

Microbore 0.5, 1 & 2 mm ~5 – 1000 ul/min

Analytical 4.6 mm ~500 – 5000 ul/min

Particle parameters • resin diameter: e.g., 5 um silica particles • pore size: e.g., 200 Angstroms • stationary phase: e.g., C18 (octadecylsilyl) • solid support: e.g., silica

SUPPLEMENTAL: Chromatography Reference Slide


Capillary Downscale Factor Increase in Sensitivity as Column Diameter Decreases

Since Mass Spec is a Concentration-dependent Technique

Concentration Factor =

d = column Internal Diameter (ID), mm

dconventional

dmicro

2

Capillary LC =

Nano LC =

4.6

0.3

2

= 235x

4.6

0.075

2

= 3760x



Reference: Capillary Downscale Factor Example of concentrating effect of cap LC relative to narrowbore LC

GOAL: Concentrate analyte of interest for best Mass Spec detection (increase Signal to Noise ratio)

OUTCOME: Detection of molecules at attomole and femtomole amounts of material

2 1.0 mm ID

0.075 mm ID = 178x

2.6 uM 0.012 uM

= 216 ≈

Exp A: Exp B:

Column Inner Diameter (ID, mm)

1 0.075

Amt of Peptide Injected 200 fmol 200 fmol

Column Flowrate 50 ul/min 0.3 ul/min

Peak elution time for Peptide A

20 seconds 15 seconds

Peptide eluate volume 16,666 nl 75 nl

Peptide concentration in elution volume

0.012 uM 2.6 uM



Minutes

0 10 20 30 40 50 60 70 80 90

mA

U

0

2

4

6

8

10

12

14

16

mA

U

0

2

4

6

8

10

12

14

16

UV2

112311VivekMath_CtrlandCase_highPHc18stepGrad 2 Dimensional LC-MS performance depends on:

1. peak capacity in both chromatographic dimensions

2. separation orthogonality

• Column 1 Elution Profile • Collect peptides in 2 minute intervals with UV detector and

fraction collector

Column 2: LC-MS Peptide Elution Profiles

Mas

s Sp

ec A

bso

lute

Inte

nsi

ty

2 minute intervals

Time (sec)

Inte

nsity,

mA

U (

214 n

m)


Typical Numbers of Proteins Identified: • 1D LC-MS: up to 300 • 2D LCMS: up to 3000 (sample and dynamic range dependent)


RT: 0.67 - 73.58

5 10 15 20 25 30 35 40 45 50 55 60 65 70

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

dan

ce

39.08

28.5642.32 46.09 53.64

49.6159.65

24.4532.33 54.1934.17

57.26

17.92 62.4422.91 63.78

68.3717.6713.801.63 4.17

NL: 8.15E8

TIC F: FTMS + c NSI Full ms [360.00-1800.00] MS georg_hamel_011713_12503_chymo_tubA_025dda

Next slide: see Full Scan/Survey Scan at 39 minutes

Retention time 39 minutes

1 Dimensional LC-ESI-MS Elution Profile


georg_hamel_011713_12503_chymo_tubA_025dda #4335 RT: 39.22 AV: 1 NL: 5.65E7F: FTMS + c NSI Full ms [360.00-1800.00]

500 600 700 800 900 1000 1100 1200 1300 1400

m/z

0

10

20

30

40

50

60

70

80

90

100

Rela

tive A

bundance

851.95

782.40671.68

587.05553.61829.91

504.01

1173.091007.02739.37616.32 929.73

689.84

1136.261063.51

863.44

1239.31 1350.02 1427.47445.12

666.34

477.43

• Mass Spectrum at 39.22 minutes shows co-eluting peptides

MS Data Acquisition • MS1 spectrum (below) • Peaks below represent INTACT peptide m/z values


georg_hamel_011713_12503_chymo_tubA_025dda #4335 RT: 39.22 AV: 1 NL: 5.65E7F: FTMS + c NSI Full ms [360.00-1800.00]

500 600 700 800 900 1000 1100 1200 1300 1400

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

dan

ce

851.95

782.40671.68

587.05553.61829.91

504.01

1173.091007.02739.37616.32 929.73

689.84

1136.261063.51

863.44

1239.31 1350.02 1427.47445.12

666.34

477.43

1

2 3 4 5 6

• Program MS2 acquisition (MS/MS) on the Top 6 most intense peaks

• Repeat cycle: full scan + 6 MS2 (during entire LC-MS analysis)

MS Data Acquisition • MS1 spectrum (below) • Perform ‘Top 6 Data Dependent’ Acquisition


• Select Precursor Peptide • Isolate Precursor ions in Collision Cell • Fragment precursor ions with Collision

Induced Dissociation (CID) • Measure m/z of Product ions

Tandem Mass Spectrometry


+LLLYSSQICK+

Collision Cell Quadrupole Mass Filter

+ LLL +LLLY

+LLLYS CK+

QICK+ SSQICK+

Collision Induced Dissociation 1) Electric potential applied to collision cell 2) Ions accelerated to high kinetic energy 3) Ions collide with neutral gas molecule 4) Kinetic energy is converted to internal energy 5) Chemical bond breakage occurs

Measure m/z values of product ions

+LLLYSSQICK+

N2 collision gas

N2

N2 N2

Tandem Mass Spectrometry 1st measurement (MS1) = Intact Peptide m/z 2nd measurement (MS2) = Peptide fragment ion m/z

values

PRECURSOR ion 612.8 m/z (LLLYSSQICK) filtered in Q1

Q1 q2 Detector

Collision Induced Dissociation


matrixscience.com

peptide bonds

• Peptide Backbone has 3 Bond Types (peptide bond is the weakest of the 3 bonds) • Bond Breakage Yields Complimentary Ion Types, e.g., b- and y-type

Predictable Fragment Ions Types from Peptide Dissociation


MQIFVKTLTK; MWmono = 1208.7077

m/z b series y series m/z 1062.6 MQIFVKTLT K 147.1 961.6 MQIFVKTL TK 248.1 848.5 MQIFVKT LTK 361.3 747.4 MQIFVK TLTK 462.3 619.3 MQIFV KTLTK 590.4 520.3 MQIF VKTLTK 689.5 373.2 MQI FVKTLTK 836.5 260.1 MQ IFVKTLTK 949.6 132.0 M QIFVKTLTK 1077.7

Peptide Tandem Mass Spec (MS/MS) Fragment Ions and mass values


Peptide MS/MS Spectrum Precursor [M + 2H]+2 = 698.86 m/z

hegem007_fanx0092_120812_12508_seed_soluble #3757 RT: 36.52 AV: 1 NL: 3.74E3T: FTMS + c NSI d Full ms2 [email protected] [111.00-1410.00]

200 300 400 500 600 700 800 900 1000 1100 1200 1300

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

708.37

894.47175.12

508.29

807.44263.10

300.16 995.52

392.19

637.33 1082.55

1283.63337.11

1196.59

229.12

1046.53463.16 951.49550.16

hegem007_fanx0092_120812_12508_seed_soluble #3755 RT: 36.50 AV: 1 NL: 1.11E5T: FTMS + c NSI Full ms [360.00-1800.00]

698.0 698.2 698.4 698.6 698.8 699.0 699.2 699.4 699.6 699.8 700.0 700.2 700.4 700.6

m/z

0

10

20

30

40

50

60

70

80

90

100

Rela

tive A

bundance

698.86z=2

699.36z=2

699.86z=2

Full scan 698.86 m/z precursor isotope series: z = 2

Charge state information is calculated from full scan


MS2 Spectrum and Confirmed Peptide Match +TOF Product (612.8): Experiment 2, 35.118 min from bruce w 2-5 humJak3 tryp.wiffa=3.56742302613418490e-004, t0=4.31796646529983260e+001

Max. 2116.0 counts.

100 200 300 400 500 600 700 800 900 1000 1100 1200m/z, amu

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2116

Inte

ns

ity

, c

ou

nts

199.1716

86.0972307.1400

136.0725

249.1534722.3468340.2637

147.1153 420.2225885.3852 998.4837277.1471

289.1373 635.3242

a1

a2

y8(2+)

b2

y9

b3

y8

a4b4

y7

a5

y6

y5

y4

y3

y2

a3-NH3(2+) y7(2+)

y1

L L L Y S S Q I Ccam K Protein ID: Tyrosine-protein kinase JAK3 (human)


Peptide Tandem MS can provide Unambiguous Evidence for Location of

Amino Acid Modifications

Rule: Experimental data must contain fragment ions that

provide site localization evidence


MQIFVKTLTK MWmono = 1208.7077 MQIFVKTpLTK MWmono = 1288.6740 (phosphorylation +80 Da = HPO3)

Mass shifts will occur only in fragments containing the phos-Thr, therefore location of MODIFICATION can be pinpointed


Tandem MS (or MS/MS) for Identification of Amino Acid Post-translational Modification (PTM) Site


+80

+80

+80

+80

+80

+80

+80

+80

+80


Mass shifts will occur only in fragments containing the phos-Thr, therefore location of MODIFICATION can be pinpointed

Predictable Mass Shifts for Phosphorylated Fragments MQIFVKTpLTK


m/z b series y series m/z 1142.6 MQIFVKTpLT K 147.1 1041.5 MQIFVKTpL TK 248.1 928.4 MQIFVKTp LTK 361.3 747.4 MQIFVK TpLTK 542.3 619.3 MQIFV KTpLTK 670.4 520.3 MQIF VKTpLTK 769.4 373.2 MQI FVKTpLTK 919.5 260.1 MQ IFVKTpLTK 1029.6 132.0 M QIFVKTpLTK 1157.6

MQIFVKTpLTK (+80 = HPO3); MWmono = 1288.67

Mass shifts will occur only in fragments containing the phos-Thr (bold), therefore location of MODIFICATION can be pinpointed

Predictable Mass Shifts for Phosphorylated Fragments

Documents

Outline Terminology - University of Minnesota · PDF file•Detergent and salt free solution ... pigments were passed through calcium carbonate using petroleum ether ... Nano LC =